CN113698555A - PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof - Google Patents

PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof Download PDF

Info

Publication number
CN113698555A
CN113698555A CN202010438370.9A CN202010438370A CN113698555A CN 113698555 A CN113698555 A CN 113698555A CN 202010438370 A CN202010438370 A CN 202010438370A CN 113698555 A CN113698555 A CN 113698555A
Authority
CN
China
Prior art keywords
polymer
drug conjugate
drug
dtx
peg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010438370.9A
Other languages
Chinese (zh)
Inventor
汪贻广
杜洪亮
赵绥
王增辉
王瑶琪
张强
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University
Original Assignee
Peking University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University filed Critical Peking University
Priority to CN202010438370.9A priority Critical patent/CN113698555A/en
Publication of CN113698555A publication Critical patent/CN113698555A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2/00—Processes of polymerisation
    • C08F2/38—Polymerisation using regulators, e.g. chain terminating agents, e.g. telomerisation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur
    • C08G65/3348—Polymers modified by chemical after-treatment with organic compounds containing sulfur containing nitrogen in addition to sulfur
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Polymers & Plastics (AREA)
  • Dispersion Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention provides a series of polymer-drug conjugates with pH/cathepsin B step-by-step response characteristics, micelles, and a preparation method and application thereof. The polymer-drug conjugate consists of a hydrophilic chain segment and a hydrophobic chain segment, wherein the hydrophilic chain segment mainly consists of strong hydrophilic polymers such as polyethylene glycol, the hydrophobic chain segment consists of polyalkylaminoacrylate units with tertiary amino structures shown in a structural formula 1 and drug precursor units with Gly-Phe-Leu-Gly polypeptide connecting arms, and symbols are defined in the specification. In addition, the present invention also provides the use of the polymer-drug conjugate, micelle in chemotherapy of tumor and inflammatory diseases and in combination with immunotherapy and photodynamic therapy.
Figure DDA0002503155910000011

Description

PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a polymer-drug conjugate, a micelle and a micelle composition with pH/cathepsin B step-by-step response drug release characteristics, and a preparation method and medical application thereof.
Background
Nano-drugs are novel drug delivery technologies developed based on the disciplines of nanotechnology, material chemistry, pharmaceutics, clinical medicine, and the like, and are now widely used in the prevention, diagnosis, and treatment of various diseases. Through the nano design of the medicine, the in-vivo pharmacokinetic characteristics and tissue distribution of the medicine can be obviously improved, the delivery efficiency and the treatment efficacy of the medicine are improved, and the nano-design of the medicine has great potential in clinical research. The polymer-drug conjugate is a drug delivery system formed by connecting drug molecules with polymer macromolecules through chemical covalent bonds, and belongs to the field of nano drug delivery and the category of polymer therapy. Compared with the physical coating of the traditional nano-drug, the polymer-drug combination has better stability in the delivery process, and can effectively avoid the early burst release of the drug and improve the delivery controllability. If hydrophobic drug molecules are connected to hydrophilic polymer macromolecules, the water solubility of the hydrophilic polymer molecules can be greatly improved, the hydrophilic polymer molecules are prevented from being removed by body conditioning proteins, and the blood circulation time of the hydrophilic polymer molecules is prolonged. In addition, the hydrophobic drugs can be wrapped in the micelle cores in the self-assembly micellization process of the amphiphilic polymer, so that the stimulation and degradation of the external environment are effectively avoided.
The nanoscale polymer-drug conjugate can be passively targeted to a tumor part through the EPR effect of tumor tissues, so that the concentration of the drug in the tumor tissues is increased. However, many studies have also shown that such an increase in local drug concentration does not result in a significant increase in efficacy. The great resistance of the nano-drug delivered in the deep part of a complex tumor microenvironment causes the nano-drug to have poor infiltration and distribution capacity on solid tumor tissues, thus greatly limiting the clinical transformation of the nano-drug. In addition, other studies show that nanoparticles with ultra-small particle size (<6nm) show strong permeability and distribution capability to solid tumor tissues due to small diffusion resistance, but the ultra-small nano-sized polymer-drug conjugate has fast clearance rate in vivo and poor accumulation capability to target tissues, and cannot fully exert the therapeutic advantages of nano-drugs. Therefore, how to utilize the unique microenvironment of the focus part and combine the delivery advantages of nano-drugs with different particle size ranges to develop a novel polymer-drug combination system has important scientific significance for improving the drug delivery efficiency and the treatment efficacy and reducing the toxic and side effects of the drugs.
In recent years, more and more researchers begin to apply an intelligent response type drug delivery technology to the design and development of polymer-drug conjugates, construct a polymer-drug conjugate system with an endogenous or exogenous intelligent "on-off" effect, so that the conjugate system can maintain sufficient stability in blood circulation, improve blood circulation time and tumor accumulation, change physicochemical properties of the conjugate system accumulated in tumor tissues under the action of endogenous or exogenous stimulating factors, improve the penetration and distribution of the conjugate system to the deep part of a tumor, and achieve the purpose of improving drug effect. However, the smart responsive polymer-drug conjugate system reported at present has poor signal responsiveness to diseases in vivo and weak permeation-promoting ability, and it is required to develop a novel smart responsive polymer-drug conjugate system having a faster signal response speed and stronger permeation-promoting ability.
The inventors have conducted extensive research to overcome the drawbacks of the prior art and overcome the dual barrier of drug delivery both extracellular and intracellular, and as a result, have accomplished the present invention.
Disclosure of Invention
The invention provides a series of pH/cathepsin B gradual response polymer-drug conjugates with rapid acid response dissolution penetration promotion effect and high-efficiency intracellular drug release capability, micelles and micelle compositions prepared by the conjugates, and preparation methods and medical applications thereof.
In the polymer-drug conjugate, drug molecules (such as chemotherapeutic drugs) are covalently linked to a hydrophobic chain segment of a conjugate skeleton through a Gly-Phe-Leu-Gly polypeptide linking arm. Under normal physiological conditions, drug molecules can be hidden in the inner core of the micelle through self-assembly of the polymer and the drug, so that degradation by organisms and toxic and side effects on normal tissues are avoided, and the step-by-step response release of the pH/cathepsin B of the drug in extracellular and intracellular states is specifically realized in diseased tissues, so that a small molecule drug delivery barrier is overcome, and the delivery efficiency and the treatment efficacy are improved. The polymer-drug conjugates of the present invention provide a safer and more reliable drug option for chemotherapy of tumors and inflammatory diseases. In addition, the conjugates can also be used in combination with immunotherapy and photodynamic therapy to enhance their therapeutic efficacy.
The present invention aims to provide a polymer-drug conjugate having a pH/cathepsin B stepwise responsive drug release characteristics.
The polymer-drug conjugate provided by the invention consists of a hydrophilic chain segment and a hydrophobic chain segment.
Wherein the hydrophilic chain segment is selected from one or more of polyethylene glycol, polyethylene oxide, poly (N- (2-hydroxypropyl) methacrylamide), polyvinylpyrrolidone and polymethyl acrylate phosphatidyl choline.
Wherein, the hydrophobic chain segment has a structure shown in a structural formula 1:
Figure BDA0002503155890000021
wherein R ', R', X1，X2Are respectively selected from-H, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 naphthenic base and C1-C12 substituted naphthenic base;
r "" is a polymerization-derived end group selected from a thiol, thioester, alkyl, cycloalkyl, aromatic, or an end-group substituent selected from a fluorescent molecule, a photothermal probe, or a metal chelating group.
R1、R2、R3、R4The four groups can be the same or different and are respectively selected from C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aryl, C1-C16 heteroaryl and the substituent groups;
a and b are respectively integers of 1-10;
x and y are integers, and the sum of the x and the y is an integer of 20-200;
z is an integer of 1-10, and the three units x, y and z can be combined and arranged in any order;
f is an active drug molecule, and each F may be different.
Wherein R ', R ", R'" may be the same or different and are each C1-C6 alkyl; x1，X2Are each hydrogen; r "" is trithio ester.
R1、R2、R3、R4May be the same or different and are each a C1-C6 alkyl group; a and b are respectively integers of 1-5; x and y are each an integer, the sum of whichIs an integer of 60 to 100; the other symbols are as described above.
Wherein the active drug molecule is selected from the group consisting of chemotherapeutic drugs, photosensitizers, fluorescence quenchers, immunotherapeutic drugs, and photothermal probes.
Wherein the active drug molecule is a chemotherapeutic drug.
The polymer-drug conjugate provided by the invention has a structure shown in a structural formula 2:
Figure BDA0002503155890000031
wherein, Y1Can be selected from-H, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 cycloalkyl, C1-C12 substituted cycloalkyl, carboxyl and active ester group thereof, metal chelating group and maleimide ester group;
n is an integer of 5 to 500;
other symbols are as indicated above.
Preferably, wherein Y is1Is C1-C6 alkyl; n is an integer of 50 to 150; other symbols are as indicated above.
Preferably, the polymer-drug conjugate according to the present invention has a structure represented by structural formula 3:
Figure BDA0002503155890000032
Figure BDA0002503155890000041
wherein R is1’、R2' is selected from the following structures:
Figure BDA0002503155890000042
r "" is trithio ester.
The sum of x and y is 70-90;
f is as defined above.
Wherein the active drug molecule is selected from the group consisting of taxanes and non-taxane chemotherapeutic drugs.
It is another object of the present invention to provide a method for synthesizing a polymer-drug conjugate.
The invention relates to a method for synthesizing a polymer-drug conjugate, which comprises the following steps: synthesizing polymer-drug conjugate macromolecules by using a RAFT polymerization technology and modifying the end group of the RAFT polymer.
It is another object of the present invention to provide polymer-drug conjugate micelles.
The polymer-drug conjugate micelle of the present invention is formed by one or more polymer-drug conjugates in an aqueous medium through a self-assembly process.
The polymer-drug conjugate micelle of the present invention comprises a polymer-drug conjugate in which the active drug is a chemotherapeutic drug.
The polymer-drug conjugate micelle provided by the invention is characterized in that a chemotherapeutic drug is docetaxel DTX.
The micelle composition of the present invention comprises polymer-drug conjugate micelles.
It is another object of the present invention to provide the use of the polymer-drug conjugate in the preparation of a medicament for the treatment of malignant tumors and inflammatory diseases.
The polymer-drug conjugate micelle is applied to the preparation of drugs for treating malignant tumors and inflammatory diseases.
The polymer-drug conjugate micelle disclosed by the invention is applied to preparation of drugs for treating malignant tumors and inflammatory diseases when being combined with other drugs.
Compared with the prior art, the invention has the following advantages:
1. the pH/cathepsin B step-by-step response polymer-drug conjugate micelle provided by the invention has the rapid acid response dissolution and penetration promotion effect of a tumor microenvironment and high-efficiency intracellular drug release capability, can effectively overcome double obstacles of drug delivery in vitro and in vivo, realizes accurate intracellular delivery of active drugs, and realizes effective intracellular release.
2. The polymer-drug conjugate micelle provided by the invention can keep enough stability in blood circulation and normal tissues, avoids drug release and effectively reduces the toxic and side effects of the drug on the normal tissues; and the rapid response to the slightly acidic environment is realized in the diseased tissue, the micelle is dispersed to promote the penetration, and the drug delivery efficiency and the treatment efficacy are improved.
3. The synthetic method is simple and reliable, and can be combined with various treatment strategies to realize the synergistic antitumor effect. For example, chemotherapy is combined with immunotherapy, chemotherapy is combined with photothermal therapy, and the like.
Drawings
FIG. 1 shows high performance liquid chromatography (A) and hydrogen nuclear magnetic resonance (B) spectra of the polymer-drug conjugate PEG-PC7A-DTX synthesized in example 2.
FIG. 2 shows high performance liquid chromatography (A) and NMR spectra (B) of the polymer-drug conjugate PEG-PEPA-DTX synthesized in example 2.
FIG. 3 shows HPLC (A) and NMR (B) of the polymer-drug conjugate PEG-PDBA-DTX synthesized in example 2.
FIG. 4 is a high performance liquid chromatography and NMR spectra of the polymer-drug conjugates synthesized in example 2, PEG-PCHA-DTX (A, B), PEG-PDPA-DTX (C, D), PEG-iDPA-DTX (E, F) and PEG-PEHA-DTX (G, H).
FIG. 5 is a high performance liquid chromatography of the polymer-drug conjugate PEG-PC7A-DTX synthesized in example 2 before and after papain cleavage.
FIG. 6 is a graph showing UV absorption spectra (A) of Cy5-PEG-PC7A-DTX and Cy5-PEG-PCHA-DTX of the polymer-drug conjugate synthesized in example 3, fluorescence absorption ratio changes (B) of Cy5-PEG-PC7A-DTX micelles and Cy5-PEG-PCHA-DTX micelles prepared in example 4 according to changes in pH values, and fluorescence emission spectra (D) of Cy5-PEG-PC7A-DTX micelles (C) and Cy5-PEG-PCHA-DTX micelles (D) at pH 7.4 and pH 6.6.
FIG. 7 is a graph (A) showing changes in particle size distribution and a graph (B) showing changes in potential of PEG-PC7A-DTX micelles and PEG-PCHA-DTX micelles prepared in example 4 under the conditions of pH 7.4 and pH 6.6.
FIG. 8 is an electron micrograph of PEG-PC7A-DTX micelles and PEG-PCHA-DTX micelles prepared in example 4 under pH 7.4 and pH 6.6 conditions.
FIG. 9 is a graph of cumulative DTX release (%) -time (h) in different media for PEG-PC7A-DTX micelle (A) and PEG-PCHA-DTX micelle (B) prepared in example 4.
FIG. 10 is a CCK-8 method for examining the cytotoxic effect of PEG-PC7A-DTX micelles and PEG-PCHA-DTX micelles prepared in example 4 on 4T1 cells under incubation conditions of pH 7.4(A) and pH 6.6 (B).
FIG. 11 is a graph of the uptake of Cy 5-labeled PEG-PC7A-DTX micelles and PEG-PCHA-DTX micelles prepared in example 4 in 4T1 cells under incubation conditions of pH 7.4 and pH 6.6, which were examined by flow cytometry.
Fig. 12 is a graph of the study of lysosome co-localization in 4T1 cells of Cy 5-labeled PEG-PC7A-DTX micelles prepared in example 4 under incubation conditions of pH 7.4 and pH 6.6 using confocal laser microscopy, scale bar: 25 μm.
FIG. 13 is a graph of the permeation capacity of Cy 5-labeled PEG-PC7A-DTX micelles and PEG-PCHA-DTX micelles prepared in example 4 under the incubation conditions of pH 7.4 and pH 6.6 to 4T1 cell tumor spheres in vitro, which is examined by the confocal laser microscopy slicing technique, and the proportions are as follows: 100 μm. .
FIG. 14 is the in vivo anti-tumor effect of PEG-PC7A-DTX micelles and PEG-PCHA-DTX micelles prepared in example 4, the commercially available prescription Tasotrie and blank micelles PEG-PC7A and PEG-PCHA on the BALB/c mouse model of 4T1 tumor at the animal level.
FIG. 15 is a graph of the combined anti-tumor effect of PEG-PC7A-DTX micelles prepared in example 4 and the immune checkpoint inhibitor α -PD-1 on a C57BL/6 mouse model of B16 OVA-bearing tumors at the animal level.
Detailed Description
The invention provides a polymer-drug conjugate with pH/cathepsin B gradual response drug release characteristics, which consists of a hydrophilic chain segment and a hydrophobic chain segment.
Wherein the hydrophilic segment of the polymer-drug conjugate is selected from one or more of polyethylene glycol, polyethylene oxide, poly (N- (2-hydroxypropyl) methacrylamide), polyvinylpyrrolidone and polymethyl acrylate phosphatidylcholine, preferably polyethylene oxide or polyethylene glycol.
The hydrophobic segment of the polymer-drug conjugate has a structure shown in formula 1:
Figure BDA0002503155890000061
wherein R ', R', X1，X2Are respectively selected from-H, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 naphthenic base and C1-C12 substituted naphthenic base; r ', R ", R'" are preferably C1-C6 alkyl, more preferably-CH3，X1，X2preferably-H.
R "" is a polymerization-derived end group selected from a thiol, thioester, alkyl, cycloalkyl, aromatic, or an end-group substituent, preferably a thioester. In some embodiments, R "" is a fluorophore, e.g., Cy5, Cy7.5, or TAMRA.
R1、R2、R3、R4The four groups are respectively selected from C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aryl, C1-C16 heteroaryl and the above substituent groups, preferably C1-C6 alkyl, more preferably C2-C4 alkyl, the four groups can be the same or different and can be straight-chain alkyl such as ethyl, propyl and butyl or branched-chain alkyl such as isopropyl.
a and b are respectively integers of 1-10, preferably 1-5, and more preferably 2;
x and y are integers, the sum of the x and the y is an integer of 20-200, the sum of the x and the y is preferably 60-150, and more preferably 70-90;
z is an integer of 1 to 10, preferably 1 to 5, more preferably 2 to 3;
the three units of x, y and z can be combined and arranged in any order;
f is an active drug molecule selected from the group consisting of chemotherapeutic drugs, photosensitizers, fluorescence quenchers, immunotherapeutic drugs, and photothermal probes. Each F may be different.
Wherein the chemotherapeutic drug is selected from one or more of docetaxel, paclitaxel, dorubicin, epirubicin, camptothecin and hydroxycamptothecin, and preferably taxane drugs such as docetaxel and paclitaxel.
The photosensitizer is selected from porphyrin photosensitizers such as chlorin, pyropheophorbide a, verteporfin, photo-cloro and mono-asparaginyl porphin, and non-porphyrin photosensitizers such as cationic photosensitizer, quinone photosensitizer and curcumin photosensitizer.
The fluorescence quenching agent is selected from QSY, QXL, DABCYL and DABSYL.
The immunotherapy drug is selected from toll-like receptor agonist, such as CpG, R848, or small molecule IDO inhibitor.
The photothermal probe is selected from small molecule probes such as ICG, IR780, IR783, Cy7.5, IR808 and the like.
In some embodiments, the polymer-drug conjugates provided herein have a structure as shown in structural formula 2:
Figure BDA0002503155890000071
wherein, Y1Can be selected from-H, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 cycloalkyl, C1-C12 substituted cycloalkyl, carboxyl and active ester groups thereof, metal chelating groups and maleimide ester groups, and preferably C1-C6 branched alkyl.
n is an integer of 5 to 500, preferably 112;
other symbols are shown in formula 1.
In some embodiments, the polymer-drug conjugates provided herein have a structure as shown in structural formula 3:
Figure BDA0002503155890000081
wherein R is1’、R2' is selected from the following structures:
Figure BDA0002503155890000082
other symbols are shown in formula 1.
The invention also provides a synthesis method of the polymer-drug conjugate, which comprises the synthesis of polymer-drug conjugate macromolecules by using an RAFT polymerization technology and an RAFT polymer end group modification technology.
For example, the polymer-drug conjugates of the present invention can be synthesized by:
the method comprises the following steps: synthesis of macromolecular chain transfer agent
Figure BDA0002503155890000083
The terminal group of the RAFT micromolecule chain transfer agent is-COOH, and the terminal group is Y1The polyethylene glycol (unmodified hydroxyl group at the other end) of (2) was dissolved in methylene chloride, and then a catalytic amount of 4-dimethylaminopyridine and dicyclohexylcarbodiimide were added to the reaction solution. And after the reaction is carried out for 36 to 72 hours in a dark place at room temperature, filtering to remove precipitates, carrying out rotary evaporation and concentration on the organic filtrate, carrying out precipitation and purification on the organic filtrate for a plurality of times by using glacial ethyl ether, and drying to obtain the polyethylene glycol macromolecular chain transfer agent.
Step two: RAFT polymerization of polymer-drug conjugates
Figure BDA0002503155890000091
Mixing the polyethylene glycol macromolecular chain transfer agent with the RAFT reagent end group obtained in the step one with each acrylate monomer and prodrug monomer according to a specific molar ratio, dissolving the mixture by using a proper amount of N, N-dimethylformamide or 1,4 dioxane, then adding an initiator azodiisobutyronitrile with the molar equivalent of 1/2-1/10 of the polyethylene glycol macromolecular chain transfer agent, uniformly mixing, carrying out freezing-nitrogen filling-melting circulation for 3 times to remove oxygen, placing the reaction at 60-90 ℃ for reaction for 6-48 hours, dialyzing the reaction product for 48-72 hours at the end point of the reaction before N, N-dimethylformamide to remove unreacted micromolecules, dialyzing distilled water, and carrying out freeze drying to obtain the polymer-drug conjugate.
Wherein, X1，X2、Y1，X2、R1、R2、R3、R4R ', R', n, x, y, z, a, b, etc. are as described above.
The invention also provides a terminal fluorescence modification strategy of the polymer-drug conjugate precursor macromolecule. The fluorescent molecule is selected from: cy5, Cy7.5, TAMRA, etc., preferably Cy 5.
For example, the Cy5 end-group fluorescently modified polymer-drug conjugates of the present invention can be synthesized by:
the method comprises the following steps: terminal aminolysis of Polymer-drug conjugates
Figure BDA0002503155890000101
Weighing a certain amount of polymer-drug conjugate precursor macromolecules obtained by RAFT polymerization technology, dissolving in methanol, ultrasonically dissolving, adding n-butylamine with molar equivalent of 10-50 times and tributylphosphine with molar equivalent of 1-5 times in nitrogen atmosphere, reacting, and stirring at room temperature for 4-8 hours under nitrogen condition. After the reaction is finished, precipitating with diethyl ether, washing, and drying in vacuum to obtain the end-thiolated polymer-drug conjugate.
Step two: cy5 coupling of thiol-terminated Polymer-drug conjugates
Figure BDA0002503155890000102
And (2) dissolving the terminal sulfhydrylation polymer-drug conjugate obtained in the step one in methanol under the nitrogen atmosphere, adding 2-4 times of maleimide Cy5 and 3-6 times of triethylamine, dissolving by ultrasonic, uniformly mixing, and continuously stirring the reaction system at room temperature for 24-48 hours. Purifying the reaction solution by a Sephadex LH20 gel column (methanol is used as an eluent), and freeze-drying the product to obtain the Cy5 end group modified polymer-drug conjugate.
Wherein, X1，X2、Y1，X2、R1、R2、R3、R4R ', R', n, x, y, z, a, b, etc. are as described above.
Another aspect of the present invention provides micelles comprising one or more of the polymer-drug conjugates of the present invention formed by a self-assembly process.
The micelle of the present invention comprises a polymer-drug conjugate in which the active drug is a chemotherapeutic drug, and one or more selected from the group consisting of a photosensitizer, a fluorescence quencher, an immunotherapeutic drug and a photothermal probe. The molar ratio of the polymer-drug conjugates containing different active molecules can be 100: 1-1: 100, and the ratio can be adjusted as required.
In the micelles of the present invention, the chemotherapeutic drug may be attached to the polymer backbone by RAFT polymerization by way of prodrug monomers, and in some embodiments, the particle size of the polymer-drug conjugate micelle comprising docetaxel is about 30-50 nm.
The preparation method of the polymer-drug conjugate micelle of the present invention may employ a desolvation method, a thin film ultrasonic method, an ethanol injection method, etc., and the desolvation method is preferred.
For example, the polymer-drug conjugate micelle of the present invention can be prepared by the following steps:
the method comprises the following steps: weighing one or more polymer-drug conjugates according to a certain proportion, adding a proper amount of organic solvent, and ultrasonically dissolving for later use;
step two: taking ultrapure water with the volume ratio of 10-100, quickly adding the solution obtained in the step one into the ultrapure water under the ultrasonic condition of a probe, and continuously carrying out ultrasonic treatment for 1-3 minutes;
step three: and transferring the micellar solution containing the organic solvent after the ultrasonic treatment into an ultrafiltration tube, and carrying out ultrafiltration for 3-5 times by using ultrapure water to remove the organic solvent. And (4) centrifuging to remove insoluble parts to obtain a micelle solution.
Preferably, in the micelle of the present invention, the active small molecule is docetaxel, which is a chemotherapeutic drug.
The organic solvent described in the above "step one" in the preparation of the micelle of the present invention is preferably: tetrahydrofuran, methanol, acetonitrile and mixtures thereof, more preferably methanol; the ratio of the ultrapure water to the organic solvent in "step two" is preferably 10 to 30, more preferably 20; the duration of the ultrasound in the step two is preferably 1 to 2 minutes, more preferably 1 minute; the number of continuous ultrafiltration in "step three" is preferably 4 to 5 minutes, more preferably 4 times;
preferably, the docetaxel-containing polymer-drug conjugate micelle of the present invention may be prepared by the following steps:
the method comprises the following steps: weighing 5mg of polymer-drug combination containing docetaxel in 200 mu L of methanol, and ultrasonically dissolving for later use;
step two: putting 4mL of ultrapure water into an EP (EP) tube, quickly adding 200 mu L of the polymer-drug combination methanol solution obtained in the step one into the ultrapure water under the ultrasonic condition of a probe, and continuously performing ultrasonic treatment for 1 minute;
step three: the micellar solution after the end of sonication was transferred to a 100kD ultrafiltration tube and ultrafiltered 4 times (4000rpm,15min) with ultra pure water to remove methanol. Centrifuging at 10000rpm for 10min to remove insoluble part, and transferring the supernatant to a new EP tube to obtain the polymer-drug conjugate micelle solution containing docetaxel.
The invention also provides the medical application of the polymer-drug conjugate micelle in the field of tumor treatment.
The present invention will be described and illustrated with reference to the following specific examples, but the present invention is not limited to these specific examples.
In the following examples, unless otherwise indicated, each symbol or abbreviation represents the following meanings:
PEG-OH: monomethyl terminated polyethylene glycol
Figure BDA0002503155890000111
PEG-CTA: macromolecular chain transfer agent
Figure BDA0002503155890000112
CTA: trithioester chain transfer agents
Figure BDA0002503155890000121
C7A: the monomer structure is
Figure BDA0002503155890000122
CHA: the monomer structure is
Figure BDA0002503155890000123
EPA: the monomer structure is
Figure BDA0002503155890000124
DPA: the monomer structure is
Figure BDA0002503155890000125
iDPA: the monomer structure is
Figure BDA0002503155890000126
DBA: the monomer structure is
Figure BDA0002503155890000127
EHA: the monomer structure is
Figure BDA0002503155890000128
MA-GFLG-DTX: the monomer structure is
Figure BDA0002503155890000129
DTX: docetaxel
Figure BDA0002503155890000131
Cy 5-mal: maloylated Cy5
Figure BDA0002503155890000132
Example 1: synthesis of macromolecular chain transfer agent PEG-CTA
PEG-OH (5g, 1mmol) and an equivalent molar equivalent of CTA were precisely weighed in a round-bottomed flask, 50mL of dichloromethane was added, ultrasonic dissolution was performed, and then 0.1-fold molar equivalent of 4-dimethylaminopyridine and 1.5-fold molar equivalent of dicyclohexylcarbodiimide were added to the reaction solution. After 36 hours of reaction at room temperature in the dark, the precipitate was removed by ultrafiltration, and the organic filtrate was concentrated to 10mL with a rotary evaporator. The product was purified 3 times in glacial ethyl ether to give 4.2g of the macromolecular chain transfer agent PEG-CTA in 84% yield.
Example 2: RAFT polymerization and structural characterization of Polymer-drug conjugates
1. Synthesis of Polymer-drug conjugate PEG-PC7A-DTX
PEG-CTA (50mg, 0.01mmol) from "example 1" was weighed into a reaction flask, 100 molar equivalents of C7A monomer and 5 molar equivalents of MA-GFLG-DTX prodrug monomer were added, dissolved in 1mL of N, N-dimethylformamide, followed by 0.333 molar equivalents of the initiator azobisisobutyronitrile, and sonicated to dissolve it well and mix well. Freezing, filling nitrogen, melting, circulating for 3 times, removing oxygen, and reacting at 60 ℃ for 36 hours. After the reaction is finished, transferring the reaction solution into a dialysis bag of 10kD, dialyzing with N, N-dimethylformamide for 48 hours to remove unreacted monomers and other small molecules, dialyzing with distilled water for 12 hours to remove organic solvent, and freeze-drying the dialyzed product to obtain the polymer-drug conjugate PEG-PC 7A-DTX. The purified PEG-PC7A-DTX HPLC and NMR are shown in FIG. 1, the polymer liquid phase diagram is shown as a single peak in FIG. 1A, which shows that the conjugate has good purity and no free drug, and the number of C7A monomers on PEG-PC7A-DTX is 70 and the DTX drug-loading rate is 6.5% as calculated in FIG. 1B.
2. Synthesis of Polymer-drug conjugate PEG-PEPA-DTX
PEG-CTA (50mg, 0.01mmol) from "example 1" was weighed into a reaction flask, 100 times molar equivalent of EPA monomer and 5 times molar equivalent of MA-GFLG-DTX prodrug monomer were added, dissolved in 1mL of N, N-dimethylformamide, followed by 0.333 times molar equivalent of initiator azobisisobutyronitrile, and mixed well by sonication. Freezing, filling nitrogen, melting, circulating for 3 times, removing oxygen, and reacting at 60 ℃ for 36 hours. And after the reaction is ended, transferring the reaction solution into a dialysis bag of 10kD, dialyzing with N, N-dimethylformamide for 48 hours to remove unreacted monomers and other small molecules, dialyzing with distilled water for 12 hours to remove the organic solvent, and freeze-drying the dialyzed product to obtain the polymer-drug conjugate PEG-PEPA-DTX. The purified PEG-PEPA-DTX high performance liquid chromatography and nuclear magnetic resonance hydrogen spectrum are shown in figure 2, a polymer liquid phase diagram is shown to be a single peak in figure 2A, the purity of the conjugate is good, no free drug exists, the number of EPA monomers on the PEG-PEPA-DTX is calculated to be 90 from figure 2B, and the drug loading of DTX is 8.0 percent.
3. Synthesis of Polymer-drug conjugate PEG-PDBA-DTX
PEG-CTA (50mg, 0.01mmol) from "example 1" was weighed into a reaction flask, 100-fold molar equivalent of DBA monomer and 5-fold molar equivalent of MA-GFLG-DTX prodrug monomer were added, dissolved in 1mL of N, N-dimethylformamide, followed by 0.333-fold molar equivalent of initiator azobisisobutyronitrile, and mixed well by sonication. Freezing, filling nitrogen, melting, circulating for 3 times, removing oxygen, and reacting at 60 ℃ for 36 hours. And after the reaction is ended, transferring the reaction solution into a dialysis bag of 10kD, dialyzing with N, N-dimethylformamide for 48 hours to remove unreacted monomers and other small molecules, dialyzing with distilled water for 12 hours to remove the organic solvent, and freeze-drying the dialyzed product to obtain the polymer-drug conjugate PEG-PDBA-DTX. The purified PEG-PDBA-DTX high performance liquid chromatography and nuclear magnetic resonance hydrogen spectrum are shown in figure 3, a polymer liquid phase diagram is shown to be unimodal in figure 3A, the purity of the conjugate is good, no free drug exists, the number of DBA monomers on the PEG-PDBA-DTX is calculated to be 90 from figure 3B, and the drug loading of DTX is-5.0%.
4. This example simultaneously synthesizes the following polymer-drug conjugates according to the RAFT polymerization method described above: PEG-PCHA-DTX, PEG-PDPA-DTX, PEG-iDPA-DTX, PEG-PEHA-DTX. The hplc and nmr hydrogen spectra of each polymer-drug conjugate are shown in fig. 4. From fig. 4, it can be seen that the polymer liquid phase diagrams are all unimodal, free of free drug, indicating good conjugate purity. The nuclear magnetic resonance hydrogen spectrum of each polymer-drug conjugate determines that the connection number of monomers such as PEG-PCHA-DTX, PEG-PDPA-DTX, PEG-iDPA-DTX, CHA, DPA, iDPA, EHA and the like of the PEG-PEHA-DTX is 70, 90 and 90 respectively, and the DTX drug loading of the corresponding polymer-drug conjugate is about 7.0 percent, 6.0 percent, 9.0 percent and 9.0 percent respectively.
5. The ability of the polymer-drug conjugate to release free drug was determined by papain degradation, and the free DTX released from the polymer was followed by high performance liquid chromatography before and after papain cleavage, as exemplified by PEG-PC7A-DTX prepared in the above example. The result shows that through papain cleavage, PEG-PC7A-DTX can effectively release free DTX, and according to papain cleavage data, the calculated effective DTX drug loading is 5.73%, which is basically consistent with the quantitative result of nuclear magnetic resonance hydrogen spectrum, thus the method is reliable. The HPLC profiles before and after papain cleavage of the polymer-drug conjugate are shown in FIG. 5, from which it can be seen that the purity of the polymer before cleavage is good and is a single peak, while after enzymatic cleavage, a free DTX peak appears. The results all prove that the MA-GFLG-DTX prodrug monomer and the C7A monomer can be copolymerized and linked to a macromolecular chain transfer agent to form a polymer-drug conjugate, enzyme sensitive response can be realized, free DTX is released, and the polymer synthesis method is reliable.
Example 3: synthesis of Cy5 end-group fluorescent modified polymer-drug conjugate
In this example, a fluorescent molecule Cy5 was linked to the polymer through the end group of the polymer-drug conjugate, and a Cy5 end group fluorescence modified polymer-drug conjugate was synthesized.
The polymer-drug conjugate has the following structure:
Figure BDA0002503155890000151
the hydrophilic block is methyl terminated PEG (Mw ═ 5000Da), R of the side chain of the hydrophobic chain segment1' and R2"the group structure is shown below:
Figure BDA0002503155890000152
after being linked with a fluorescent molecule Cy5, the Cy5 end group fluorescence modified polymer-drug conjugate has the following structure:
Figure BDA0002503155890000153
taking polymer-drug conjugates PEG-PC7A-DTX and PEG-PCHA-DTX as examples, the end group modification method of Cy5 fluorescent molecules is carried out as follows:
synthesis of Cy5-PEG-PC7A-DTX
The method comprises the following steps: terminal aminolysis of PEG-PC7A-DTX
Weighing 100mg of polymer-drug conjugate PEG-PC7A-DTX, dissolving in methanol, ultrasonically dissolving, adding 10 times of molar equivalent of n-butylamine and 1 time of molar equivalent of tributylphosphine under nitrogen atmosphere, and reacting under nitrogen condition and stirring at room temperature for 4 hours. And clarifying the reaction solution from yellow, terminating the reaction, precipitating with diethyl ether, washing, and vacuum drying to obtain the thiol-terminated polymer-drug conjugate PEG-PC 7A-DTX-SH.
Step two: cy5 coupling of thiol-terminated Polymer-drug conjugates
Weighing 50mg of the thiol-terminated polymer-drug conjugate obtained in the first step, dissolving in 1mL of methanol under nitrogen atmosphere, adding 2 times of molar equivalent of Cy5-mal and 3 times of molar equivalent of triethylamine, dissolving by ultrasonic, mixing uniformly, and continuously stirring the reaction system for 48 hours under nitrogen atmosphere at room temperature. And purifying the reaction product by using a gel column Sephadex LH20, using methanol as an eluent, collecting a polymer part, and freeze-drying the product to obtain the Cy5 end group fluorescence modified polymer-drug conjugate Cy5-PEG-PC7A-DTX with the yield of 81 percent and the mass fraction of Cy5 fluorescent molecules in the polymer of 0.5 percent. The thin layer chromatography detection result of the product shows that the product has good purity, and the ultraviolet spectrum of the product is shown in figure 6.
Synthesis of Cy5-PEG-PCHA-DTX
The method comprises the following steps: terminal aminolysis of PEG-PCHA-DTX
Weighing 100mg of polymer-drug conjugate PEG-PCHA-DTX, dissolving in tetrahydrofuran, ultrasonically dissolving, adding 10 times of molar equivalent of n-butylamine and 1 time of molar equivalent of tributylphosphine under nitrogen atmosphere, and reacting under nitrogen condition and stirring at room temperature for 4 hours. And clarifying the reaction solution from yellow, terminating the reaction, precipitating with diethyl ether, washing, and vacuum drying to obtain the thiol-terminated polymer-drug conjugate PEG-PCHA-DTX-SH.
Step two: cy5 coupling of thiol-terminated Polymer-drug conjugates
Weighing 50mg of the thiol-terminated polymer-drug conjugate obtained in the step one, dissolving the thiol-terminated polymer-drug conjugate in 1mL of tetrahydrofuran under nitrogen atmosphere, adding 2 times of molar equivalent of Cy5-mal and 3 times of molar equivalent of triethylamine, dissolving by ultrasound, mixing uniformly, and continuously stirring the reaction system for 48 hours under nitrogen atmosphere at room temperature. And purifying the reaction product by using a gel column Sephadex LH20, taking tetrahydrofuran as an eluent, collecting a polymer part, and freeze-drying the product to obtain the Cy5 end group fluorescence modified polymer-drug conjugate Cy5-PEG-PCHA-DTX with the yield of 81% and the mass fraction of Cy5 fluorescent molecules in the polymer of 0.5%. The thin layer chromatography detection result of the product shows that the product has good purity, and the ultraviolet spectrum of the product is shown in figure 6.
Example 4: preparation of polymer-drug conjugate micelle
This example prepared various polymer-drug conjugate micelles using a desolvation method.
Preparation of PEG-PC7A-DTX micelle
Accurately weighing 5mg of the polymer-drug conjugate PEG-PC7A-DTX prepared in "example 2" in 200. mu.L of methanol, and ultrasonically dissolving for later use; adding another 4mL of ultrapure water into an EP (EP) tube, quickly adding the methanol solution of the polymer-drug combination into the ultrapure water under the ultrasonic condition of the probe, and continuing performing ultrasonic treatment for 1 minute; the micellar solution after the end of sonication was transferred to a 100kD ultrafiltration tube and ultrafiltered 4 times (4000rpm,15min) with ultra pure water to remove methanol. Centrifuging at 10000rpm for 10min to remove insoluble part, and transferring the supernatant to a new EP tube to obtain PEG-PC7A-DTX micelle solution.
Preparation of Cy5-PEG-PC7A-DTX micelles
Precisely weighing 5mg of the polymer-drug conjugate Cy5-PEG-PC7A-DTX prepared in 'example 3' in 200 μ L of methanol, and ultrasonically dissolving for later use; adding another 4mL of ultrapure water into an EP (EP) tube, quickly adding the methanol solution of the polymer-drug combination into the ultrapure water under the ultrasonic condition of the probe, and continuing performing ultrasonic treatment for 1 minute; the micellar solution after the end of sonication was transferred to a 100kD ultrafiltration tube and ultrafiltered 4 times (4000rpm,15min) with ultra pure water to remove methanol. Centrifuging at 10000rpm for 10min to remove insoluble part, transferring the supernatant to a new EP tube to obtain Cy5-PEG-PC7A-DTX micellar solution, wherein the fluorescence absorption values and fluorescence emission spectra in different pH environments are shown in FIG. 6.
Preparation of Cy5-PEG-PC7A-DTX normally bright micelle
4mg of PEG-PC7A-DTX prepared in "example 2" and 1mg of Cy5-PEG-PC7A-DTX prepared in "example 3" were precisely weighed and dissolved in 200. mu.L of methanol for use; adding another 4mL of ultrapure water into an EP (EP) tube, quickly adding the methanol solution of the polymer-drug combination into the ultrapure water under the ultrasonic condition of the probe, and continuing performing ultrasonic treatment for 1 minute; the micellar solution after the end of sonication was transferred to a 100kD ultrafiltration tube and ultrafiltered 4 times (4000rpm,15min) with ultra pure water to remove methanol. Centrifuging at 10000rpm for 10min to remove insoluble part, transferring the supernatant to a new EP tube to obtain Cy5-PEG-PC7A-DTX normally bright micelle solution.
Preparation of pH insensitive PEG-PCHA-DTX and Cy5-PEG-PCHA-DTX micelles
Respectively and precisely weighing 5mg of the polymer-drug conjugate PEG-PC7A-DTX prepared in 'example 2' or the polymer-drug conjugate Cy5-PEG-PC7A-DTX prepared in 'example 3' in 200 mu L of tetrahydrofuran, and ultrasonically dissolving for later use; adding 4mL of ultrapure water into an EP (EP) tube, and rapidly adding the polymer-drug conjugate tetrahydrofuran solutions into the ultrapure water respectively under the ultrasonic condition of a probe for 1 minute by ultrasonic treatment; the micellar solution after the end of sonication was transferred to a 100kD ultrafiltration tube and ultrafiltered 4 times (4000rpm,15min) with ultra pure water to remove tetrahydrofuran. Centrifuging at 10000rpm for 10min to remove insoluble part, transferring the supernatant to a new EP tube to obtain PEG-PCHA-DTX or Cy5-PEG-PCHA-DTX micelle solution.
5. Particle size and potential of Polymer-drug conjugate micelles
The PEG-PC7A-DTX micelle and the PEG-PCHA-DTX micelle prepared in the above examples are respectively diluted to 100 mu g/mL by buffer solutions with pH 7.4 and pH 6.6, the particle sizes and the potentials of the two micelle preparations at different pH values are measured by a dynamic light scattering instrument, and the change of the micelle morphology is observed by a transmission electron microscope. As shown in FIG. 7, the PEG-PC7A-DTX micelle showed a distinct pH sensitive property, with the particle size decreasing from-40 nm at pH 7.4 to-5 nm at pH 6.6, indicating that the micelle rapidly disintegrates with decreasing pH, showing a single polymer morphology distributed in aqueous solution. Its Zeta potential can be rapidly changed from-12.5 + -1.5 mV at pH 7.4 to a positive charge of 46.0 + -0.5 mV at pH 6.6. In contrast, the particle size and potential of the pH insensitive PEG-PCHA-DTX micelle were not much different under the two pH conditions, indicating that it did not have pH sensitive properties.
The apparent morphology of the two prodrug micelles at different pH values was further examined by transmission electron microscopy, and the results are shown in FIG. 8. The PEG-PC7A-DTX micelle shows uniform spherical shape under the condition of pH 7.4, has good dispersibility and particle size of about 30nm, and is disintegrated under the condition of pH 6.6, and the round and uniform nano-morphology completely disappears. The pH insensitive PEG-PCHA-DTX micelle shows good micelle morphology in two pH media, and the particle size is about 30 nm.
Example 5: in vitro release of polymer-drug conjugate micelles
The PEG-PC7A-DTX micelles and the PEG-PCHA-DTX micelles prepared in "example 4" were diluted to 1mg/mL with different release media (PBS 7.4, PBS 6.6, complete medium, papain solution), respectively, the samples were incubated in a constant temperature shaker at 37 ℃ and 100r/min, 50. mu.L of each sample was removed in a 1.5mL EP tube at set time points (1h, 2h, 3h, 6h, 9h, 12h, 24h, 48h), rapidly cooled in an ice bath, the samples were diluted with 150. mu.L of acetonitrile, passed through a 0.22 μm microporous membrane, and the released free drug was detected by high performance liquid chromatography. The results are shown in fig. 9, and overall, the two polymer prodrug micelles showed slow drug release behavior in PBS 7.4, PBS 6.6 and complete medium; among them, PEG-PC7A-DTX micelle was released relatively quickly. The 48h release amount of the PEG-PC7A-DTX micelle in PBS 6.6 is higher than that in PBS 7.4, while the 48h release amount of the pH-insensitive PEG-PCHA-DTX micelle in PBS 6.6 is lower than that in PBS 7.4, which is related to the pH-sensitive dissociation characteristic of the PEG-PC7A-DTX micelle. As can be seen from the drug release behaviors of the papain solution group, the PEG-PC7A-DTX micelle can release almost all the free drugs within 8h, while the 48h release amount of the drugs in the PEG-PCHA-DTX micelle is lower than 5%, which indicates that the PEG-PC7A-DTX micelle has the gradual response release characteristics to pH and cathepsin B signals.
Example 6: cytotoxic effects of Polymer-drug conjugate micelles
4T1 cells were seeded at a density of 1000 cells/well in 96-well plates at 100. mu.L/well in complete medium at 37 ℃ with 5% CO2Performing conventional culture in a constant temperature cell incubator for 24 hr, removing original culture medium after cells are completely attached to the wall, adding 100 μ L of complete culture medium containing taxol (Tasotere) with different DTX concentrations and pH 7.4 or pH 6.6 of PEG-PC7A-DTX micelle and PEG-PCHA-DTX micelle prepared in example 4 respectively to obtain final DTX concentrations of 8.663, 1.238, 0.619, 0.124, 0.062, 0.012 and 0.001 μ M, and culturing the cells at 37 deg.C and 5% CO2After incubation for 24 hours in a constant-temperature cell incubator, the culture solution containing micelles is discarded, PBS is washed once, a blank RPMI-1640 complete culture medium is added, after further culture for 24 hours, the absorbance OD value is read at 450nm on an enzyme labeling instrument (Multiskan FC, Thermo Fisher) by using a CCK-8 detection kit, the cytotoxic effect of the polymer-drug conjugate micelles on 4T1 cells under different pH values is calculated, and the result is shown in FIG. 10.
In 4T1 cells, Tasotere showed stronger cytotoxic effects than the polymer-drug conjugate micelles at different pH values, and its IC50About 10nM and a pH that has little effect on its cytotoxicity. The PEG-PC7A-DTX micelle shows stronger cytotoxic effect than the PEG-PCHA-DTX micelle under the same incubation condition, and the cytotoxic effect of the PEG-PC7A-DTX micelle under the condition of pH 6.6 is more obvious. PEG-PC7A-DTX micelleIC of 4T1 cells under incubation conditions of pH 6.650Can reach 31nM, and the in vitro cell proliferation inhibiting activity is very close to that of Tasotrie.
Example 7: cellular uptake capacity of Polymer-drug conjugate micelles
The Cy 5-labeled polymer-drug conjugate micelle prepared in "example 4" was selected to examine its uptake effect in 4T1 cells under different pH conditions. 4T1 cells in logarithmic growth phase were seeded at 40000 cells/well in 12-well plates, 1mL of complete medium per well, at 37 ℃ with 5% CO2Performing conventional culture in a constant temperature cell incubator for 24 hours, removing the original culture medium after the cells are completely attached to the wall, respectively adding 500 μ L of serum-free culture medium containing Cy5-PEG-PC7A-DTX (normally bright type) and Cy5-PEG-PCHA-DTX micelle with pH 7.4 or pH 6.6 at 37 deg.C and 5% CO2After incubation in a constant temperature cell incubator for 20 minutes, the micelle-containing culture solution was aspirated, the cells were washed twice with PBS, then trypsinized, centrifuged at 1500r/min for 5 minutes to collect the cells, washed twice with PBS, resuspended in 200 μ L PBS, and the cell uptake was detected by flow cytometry (FACS Calibur, BD), and the quantification results are shown in fig. 11. As can be seen from the cellular uptake results, the cellular uptake of the PEG-PC7A-DTX micelle at both pH values is significantly higher than that of the pH insensitive PEG-PCHA-DTX micelle, and the cellular uptake of the PEG-PC7A-DTX micelle at pH 6.6 is 5 times that of the PEG-PC7A-DTX micelle at pH 7.4. The PEG-PC7A-DTX micelle is caused by the fact that the PEG-PC7A-DTX micelle is disintegrated under the condition of pH 6.6, the particle size is reduced, and positive charges are exposed to promote the uptake, and the cell uptake of the PEG-PCHA-DTX micelle under the two pH values is not obviously different.
Example 8: intracellular lysosomal co-localization studies of polymer-drug conjugate micelles
4T1 cells in logarithmic growth phase were seeded at 40000 cells/well in a confocal dish at 37 ℃ with 5% CO2Performing conventional culture in a constant temperature cell incubator for 24 hours, removing the original culture medium after the cells are completely attached to the wall, respectively adding 500 μ L of Cy5-PEG-PC7A-DTX micelles diluted by serum-free culture media with different pH values (prepared in example 4) to make the final micelle concentration be 100 μ g/mL, and culturing the cells at 37 ℃ and 5 ℃％CO2After incubation for 20 minutes in a constant-temperature cell incubator, absorbing the culture solution containing micelles, adding a serum culture medium, continuing to culture for 2 hours, removing the culture medium, adding Hoechst 33342 working solution diluted by the serum-free culture medium, incubating for 15 minutes at 37 ℃, removing a dye solution, washing for three times by PBS, adding Lyso-Tracker Green working solution, and observing the co-localization condition of lysosomes by using a laser confocal microscope, wherein the result is shown in FIG. 12, and PEG-PC7A-DTX micelles are finally aggregated in the lysosomes under different pH values, which also provides necessary conditions for cathepsin B existing in a large amount in intracellular lysosomes to play the role of hydrolysis and fragmentation of LG polypeptide.
Example 9: research on in-vitro tumor sphere penetration capacity of polymer-drug conjugate micelle
Establishing a 4T1 mouse in-vitro tumor sphere model, when the diameter of a tumor sphere grows to 300-400 mu m, transferring the tumor sphere to an eight-hole chamber glass slide, carefully removing the original culture medium, respectively adding 300 mu L of Cy5-PEG-PC7A-DTX micelle (normally bright type) and Cy5-PEG-PCHA-DTX micelle (example 4) diluted by serum-free culture media with different pH values for 8 hours, then sucking out the culture solution containing the micelle, washing three times by PBS, fixing 30 minutes by paraformaldehyde, and washing three times by PBS. The laser confocal layer-cutting technique was used to perform transverse light-cutting at an interval of 10 μm from the top to the middle of the tumor sphere, study the fluorescence intensity of different layers of the tumor sphere, and examine the penetration ability of the preparation in the tumor sphere, the results are shown in fig. 13. Under the same fluorescence conditions, the penetration depth and the uptake intensity of the PEG-PC7A-DTX micelle under the condition of pH 6.6 are obviously higher than those under the condition of pH 7.4. And the pH insensitive PEG-PCHA-DTX micelle shows weaker fluorescence signal and poorer permeability under two pH values. The phenomenon in the in vitro three-dimensional tumor sphere proves that the PEG-PC7A-DTX micelle provided by the invention can be dispersed in a tumor microacid environment and promotes the penetration and the uptake of deep parts of tumors.
Example 10: in vivo anti-tumor effect study of polymer-drug conjugate micelle
Establishing a BALB/c mouse model of the tumor of lotus 4T1 (18-20 g female, department of laboratory animals, department of medical science of Beijing university),when the tumor volume grows to 50-100 mm3The test method is characterized in that the test method comprises the following steps of randomly dividing 6 experimental groups into 10 groups, respectively giving different administration strategies, wherein the day of the first administration is day 0, and the specific groups and the administration scheme are as follows:
1. physiological saline: respectively injecting 200 μ L physiological saline into tail vein on days 0, 2, 4, 6, and 8;
tasotere: respectively injecting 3mg/kg of Tasomite into tail vein on 0, 2, 4, 6 and 8 days;
PEG-PCHA blank micelle: respectively injecting PEG-PCHA blank micelles with corresponding doses to the PEG-PCHA-DTX drug-loaded micelles in tail vein on 0 th, 2 th, 4 th, 6 th and 8 th days;
PEG-PC7A blank micelle: respectively injecting PEG-PC7A blank micelles with corresponding doses to PEG-PC7A-DTX drug-loaded micelles into tail veins on days 0, 2, 4, 6 and 8;
PEG-PCHA-DTX micelle: respectively injecting 3mg/kg (measured by DTX) of PEG-PCHA-DTX micelle into tail vein on 0 th, 2 th, 4 th, 6 th and 8 th days;
PEG-PC7A-DTX micelle: on days 0, 2, 4, 6 and 8, 3mg/kg (measured by DTX) of PEG-PC7A-DTX micelle is injected into tail vein respectively;
from the day of administration (i.e., day 0), the tumor length (a) and the tumor length (b) of each experimental group of mice were measured with an electronic caliper every day, and the tumor volume V was a × b22 and relative tumor volume RTV ═ V/V0，V0Tumor volume at day 0. The graph of the relative tumor volume-time change is shown in fig. 14, and it can be seen that PEG-PCHA blank micelles and PEG-PCHA-DTX drug-loaded micelles have no inhibitory effect on the growth of tumors, the growth curve thereof is basically consistent with that of the normal saline control group, and PEG-PC7A blank micelles have a certain tumor-inhibiting effect during the administration period (first 12 days), but the tumors grow rapidly at the later observation period (12-17 days), and the tumor size thereof is not significantly different from that of the normal saline control group at the 17 th day. The Tasotere free drug group has obvious tumor growth inhibition effect, and the 17-day tumor inhibition rate is 32%. The PEG-PC7A-DTX micelle provided by the invention has the most obvious inhibition effect on 4T1 tumor, and can reach 73% of tumor inhibition capability. Therefore, the PEG-PC7A-DTX micelle has a good in-vivo tumor inhibition effect.
Example 11: evaluation of the synergistic antitumor Effect of PEG-PC7A-DTX micelle in combination with the immune checkpoint inhibitor alpha-PD-1
Establishing a mouse model (18-20 g female, department of medical science, department of Beijing university, laboratory animal science) of the tumor C57BL/6 of the Holo B16OVA until the tumor volume grows to 50-100 mm3The drug administration strategy is divided into 5 experimental groups at random, 7 drugs in each group are respectively given to different drug administration strategies, the day of the first drug administration is day 0, and the specific groups and the drug administration scheme are as follows:
1. physiological saline: respectively injecting 200 μ L physiological saline into tail vein on days 0, 3, 6 and 9;
2.α -PD-1: injecting alpha-PD-1 with the concentration of 75 mug/200 mug into the abdominal cavity on days 1,4, 7 and 10 respectively;
tasotere + α -PD-1: respectively injecting Tasomite with the concentration of 3mg/kg in tail vein on days 0, 3, 6 and 9, and respectively injecting alpha-PD-1 with the concentration of 75 mug/200 muL in abdominal cavity on days 1,4, 7 and 10;
PEG-PC7A-DTX micelle: on days 0, 3, 6 and 9, 3mg/kg (measured by DTX) of PEG-PC7A-DTX micelle is injected into tail vein respectively;
PEG-PC7A-DTX micelle + α -PD-1: 3mg/kg (measured by DTX) PEG-PC7A-DTX micelle is injected into tail vein on 0, 3, 6 and 9 days respectively, and 75 mug/200 mug alpha-PD-1 is injected into abdominal cavity on 1,4, 7 and 10 days respectively.
The growth of the tumor was monitored daily in each of the mice in the administration groups according to the calculation method of the relative tumor volume in "example 10", and a graph of the relative tumor volume versus time was prepared. As shown in figure 15, the B16OVA tumor growth inhibition effect is not obvious after the single intraperitoneal injection of the alpha-PD-1 for four times, and the tumor inhibition effect is obvious after the combined application of the alpha-PD-1 and the Tasomite, and the tumor inhibition rate of the 15 days is 60 percent. The PEG-PC7A-DTX micelle which is singly administrated has very obvious anti-tumor effect, and the tumor inhibition rate can reach 71 percent. The combined use of PEG-PC7A-DTX micelle and alpha-PD-1 can further enhance the anti-tumor effect, the combined tumor inhibition rate is up to 86 percent, and the anti-tumor effect is obviously different from that of a group using PEG-PC7A-DTX micelle alone and a group using Tasotrie and alpha-PD-1. Therefore, the combination of PEG-PC7A-DTX micelle and the immune checkpoint inhibitor alpha-PD-1 can exert a synergistic antitumor effect, and further enhance the antitumor effect of the PEG-PC7A-DTX micelle.
In the above experiments, the polymer-drug conjugate micelles prepared in the examples of the present invention are merely exemplary selected for illustrating the advantages of the present invention, but the present invention is not limited thereto. It is to be noted that other polymer-drug conjugate micelles of the present invention also have high drug delivery ability and exhibit superior therapeutic effects.
The present invention is described above. The invention includes within its scope various modifications of detail without departing from the scope of the claims. Moreover, all such modifications of the invention as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims (10)

1. The polymer-drug conjugate with the pH/cathepsin B gradual response drug release characteristic consists of a hydrophilic chain segment and a hydrophobic chain segment.
2. The polymer-drug conjugate of claim 1, wherein the hydrophilic segment is selected from one or more of polyethylene glycol, polyethylene oxide, poly (N- (2-hydroxypropyl) methacrylamide), polyvinylpyrrolidone and polymethylacrylate phosphatidylcholine.
3. The polymer-drug conjugate of claim 1 or 2, wherein the hydrophobic segment has a structure represented by formula 1:
Figure FDA0002503155880000011
wherein R ', R', X1，X2Are respectively selected from-H, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 naphthenic base and C1-C12 substituted naphthenic base;
r "" is a polymerization-derived end group selected from a thiol, thioester, alkyl, cycloalkyl, aromatic, or an end-group substituent selected from a fluorescent molecule, a photothermal probe, or a metal chelating group.
R1、R2、R3、R4The four groups can be the same or different and are respectively selected from C1-C16 alkyl, C1-C16 cycloalkyl, C1-C16 aryl, C1-C16 heteroaryl and the substituent groups;
a and b are respectively integers of 1-10;
x and y are integers, and the sum of the x and the y is an integer of 20-200;
z is an integer of 1-10, and the three units x, y and z can be combined and arranged in any order;
f is an active drug molecule, and each F may be different.
4. The polymer-drug conjugate of claim 3, wherein the active drug molecule is selected from the group consisting of chemotherapeutic drugs, photosensitizers, fluorescence quenchers, immunotherapeutic drugs, and photothermal probes.
5. The polymer-drug conjugate of any one of claims 1 to 4, having a structure according to formula 2:
Figure FDA0002503155880000021
wherein, Y1Can be selected from-H, C1-C12 alkyl, C1-C12 substituted alkyl, C1-C12 cycloalkyl, C1-C12 substituted cycloalkyl, carboxyl and active ester group thereof, metal chelating group and maleimide ester group;
n is an integer of 5 to 500;
the other symbols are defined according to claim 3.
6. The polymer-drug conjugate of any one of claims 1 to 5, having a structure represented by structural formula 3:
Figure FDA0002503155880000022
wherein R is1’、R2' is selected from the following structures:
Figure FDA0002503155880000031
the sum of x and y is 70-90;
f is as defined in claim 3;
r "" is trithio ester.
7. The method of synthesizing a polymer-drug conjugate of any of claims 1-5, comprising: synthesizing polymer-drug conjugate macromolecules by using a RAFT polymerization technology and modifying the end group of the RAFT polymer.
8. Polymer-drug conjugate micelles, characterized in that they are formed by one or more polymer-drug conjugates according to any of claims 1-6 by a self-assembly process.
9. A micelle composition, characterized in that it comprises the polymer-drug conjugate micelle of claim 8.
10. Use of the polymer-drug conjugate micelle of claim 8 for the preparation of a medicament for the treatment of malignant tumor and inflammatory disease.
CN202010438370.9A 2020-05-22 2020-05-22 PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof Pending CN113698555A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010438370.9A CN113698555A (en) 2020-05-22 2020-05-22 PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010438370.9A CN113698555A (en) 2020-05-22 2020-05-22 PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN113698555A true CN113698555A (en) 2021-11-26

Family

ID=78645995

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010438370.9A Pending CN113698555A (en) 2020-05-22 2020-05-22 PH/cathepsin B stepwise response polymer-drug conjugate, micelle, preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN113698555A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106995516A (en) * 2016-01-22 2017-08-01 北京化工大学 Nanometer medicine-carried system of tumour-specific enrichment and preparation method thereof
CN108066771A (en) * 2017-12-15 2018-05-25 北京思如诺科技有限公司 One kind has high drug load environmental response type anti-tumor nano drug, carrier and preparation method
CN110856750A (en) * 2018-08-16 2020-03-03 北京大学 pH-sensitive conjugate, micelle and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106995516A (en) * 2016-01-22 2017-08-01 北京化工大学 Nanometer medicine-carried system of tumour-specific enrichment and preparation method thereof
CN108066771A (en) * 2017-12-15 2018-05-25 北京思如诺科技有限公司 One kind has high drug load environmental response type anti-tumor nano drug, carrier and preparation method
CN110856750A (en) * 2018-08-16 2020-03-03 北京大学 pH-sensitive conjugate, micelle and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RUTH DUNCAN: ""THE DAWNING ERA OF POLYMER THERAPEUTICS"", 《NAT. REV. DRUG DISCOV.》, vol. 2, pages 347 - 360, XP002467298, DOI: 10.1038/nrd1088 *

Similar Documents

Publication Publication Date Title
Hu et al. Enzyme-triggered size shrink and laser-enhanced NO release nanoparticles for deep tumor penetration and combination therapy
Han et al. Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy
Wang et al. Engineering multifunctional bioactive citric acid-based nanovectors for intrinsical targeted tumor imaging and specific siRNA gene delivery in vitro/in vivo
Zhang et al. Dendrimer grafted persistent luminescent nanoplatform for aptamer guided tumor imaging and acid-responsive drug delivery
Kumar et al. Lipophilic 5-fluorouracil prodrug encapsulated xylan-stearic acid conjugates nanoparticles for colon cancer therapy
CN1318453C (en) Fluorescence labeling hydrophobic modified chitin polymer, its preparation method and application
CN108559091B (en) Polymer drug carrier with aggregation-induced emission and dual sensitivity, drug-loaded micelle and preparation method thereof
CN101501108A (en) Micelles for drug delivery
CN114377149B (en) Mn-based degradable MOF nano-reactor and preparation method and application thereof
CN105963706B (en) A kind of branching HPMA copolymer-DOX conjugate and its preparation method and application
CN111632153B (en) Chemical gene drug co-loaded targeting nano drug delivery system and preparation method thereof
Li et al. Photodynamic therapy-mediated remote control of chemotherapy toward synergistic anticancer treatment
Su et al. Enzymatic multifunctional biodegradable polymers for pH-and ROS-responsive anticancer drug delivery
Luo et al. Arginine modified polymeric micelles as a novel drug delivery system with enhanced endocytosis efficiency
CN101708335A (en) PH-sensitive dentritic polymer drug carrier
Yuan et al. Development of (G3-C12)-mediated camptothecin polymeric prodrug targeting to Galectin-3 receptor against androgen-independent prostate cancer
Tian et al. Dextran-doxorubicin prodrug nanoparticles conjugated with CD147 monoclonal antibody for targeted drug delivery in hepatoma therapy
Dai et al. A carrier-free nano-drug assembled via π–π stacking interaction for the treatment of osteoarthritis
CN112353948B (en) Drug-loaded micelle responding to acidic microenvironment to realize particle size reduction and surface charge inversion and preparation method thereof
CN107007550B (en) Redox-responsive amphiphilic copolymer and preparation method and application thereof
CN105504293A (en) Preparation and application of fluorescent star-shaped block copolymer
Hu et al. A two-photon fluorophore labeled multi-functional drug carrier for targeting cancer therapy, inflammation restraint and AIE active bioimaging
Chu et al. Stimulus‐Responsive Nano‐Prodrug Strategies for Cancer Therapy: A Focus on Camptothecin Delivery
CN114652699B (en) Size-transition type nano drug delivery carrier and preparation method and application thereof
WO2019239416A1 (en) Amphiphilic polymers, process of preparing same and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination